The specific aims including long term objectives of this project are: to isolate B12-binding proteins present in the blood plasma and blood cells (Amebocytes) of the horseshoe crab, Limulus polyphemus; to establish the primary structures of these proteins and map their B12-binding sites; to establish the biological roles of Limulus B12-binding proteins and propose mechanisms which facilitate the uptake of vitamin B12 by cells; to clone mRNA sequences encoding those Limulus proteins which bind B12 more specifically and more tightly than does the mammalian intrinsic factor. These research objectives will be achieved by using techniques related to HPLC, affinity and other types of chromatographic techniques, electrophoresis, ultra centrifugation, partial enzymatic cleavage, radiolabeling, cloning procedures, etc. Expected health related benefits of the project are: 1) Development of B12-radioassays with increased low-end sensitivity of the standard curve in the range of clinical interest. 2) Elucidation of mechanisms which facilitate the uptake of B12 by cells.